The recent development of a number of new fluorescent probes makes it possible to measure the concentrations of various intracellular free ions in single living cells. Among these ions are calcium, magnesium, sodium, potassium, and hydrogen (pH). This unit describes flow cytometric protocols using the dyes Indo-1 AM, Fluo-3, and Fura Red AM to measure intracellular calcium concentration. Support protocols detail the use of calcium buffers to calibrate a flow cytometric calcium assay, and methods to facilitate dye loading; an alternate protocol describes the use of a spectrofluorimeter to measure intracellular calcium for those investigators without access to a flow cytometer.